Fibroblast growth factor conjugates

ABSTRACT

The invention provides a conjugate comprising FGF or other polypeptide reactive with an FGF receptor, and a cytotoxic agent. The cytotoxic agent can be a ribosome-inactivating protein (RIP), such as saporin, although other cytotoxic agents can also be advantageously used. The cytotoxic agent can be attached to FGF through a chemical bond, or the composition can be prepared as a chimera using techniques of recombinant DNA. The conjugate can be used to treat FGF-mediated pathophysiological conditions by specifically targeting cells having FGF receptors and inhibiting proliferation of or causing death of such cells. Additionally, the conjugate can be used to target cytotoxic agents into cells having FGF receptors to inhibit the proliferation of such cells. The conjugate can be purified on an immobilized-heparin column.

This invention was made with Government support under Grant OK-18811awarded by the National Institutes of Health (DHHS). The Government hascertain rights in this invention.

This application is a division of U.S. Ser. No. 08/257,958, filed Jun.10, 1994, now U.S. Pat. No. 5,576,288 which is a continuation of U.S.patent application Ser. No. 08/024,682, filed Mar. 1, 1993, abandoned,which is a continuation-in-part of our U.S. patent application Ser. No.07/344,109 filed Apr. 27, 1989, entitled "Fibroblast Growth FactorConjugates", now U.S. Pat. No. 5,191,067.

This application relates to compositions which inhibit cellproliferation, and, more specifically, to fibroblast growth factorconjugated to a cytotoxic agent.

BACKGROUND OF THE INVENTION

A great deal of attention has been directed towards the identificationand characterization of factors capable of stimulating the growth andproliferation of specific cell types. In the last twenty-five years, anumber of such mitogenic factors have been isolated. Rather than havinghighly specific activities as may have been originally anticipated, manysuch growth factors are now recognized to have multifunctionalactivities, affecting a wide spectrum of cell types. In addition,certain activities are shared by homologous members of a family ofgrowth factors.

One family of growth factors now known to have a broad spectrum ofactivities is the fibroblast growth factors (FGF). Basic FGF is aprotein which has a molecular weight of approximately 16 kD, is acid andtemperature sensitive and has a high isoelectric point. A structurallyrelated protein, acidic FGF, has an acidic isoelectric point. FGFsexhibit a mitogenic effect on a wide variety of mesenchymal, endocrineand neural cells. Of particular interest is their stimulatory effect oncollateral vascularization and angiogenesis. Such mitogenic effects havestimulated considerable interest in FGF as potential therapeutic agentsfor wound healing, nerve regeneration and cartilage repair, for example.

Cells that respond to basic FGF have been shown to possess specificreceptors on the cell surface membranes. The receptor proteins appear tobe single chain polypeptides with molecular weights ranging from 110 to150 kD, depending on cell type. The proteins bind basic FGF with highaffinity (Kd=10-80 pM), with receptor numbers ranging from 2000 to80,000 per cell. The receptors can be purified from rat brain, using acombination of lectin and ligand affinity chromatography and areassociated with tyrosine kinase activity (Imamura et al., Biochem.Biophys. Res. Comm., 155:583-590 (1988); Huang and Huang, J. Biol.Chem., 261:9568-9571 (1986), both of which are incorporated herein byreference).

On baby hamster kidney cells (BHK), two basic FGF receptors withestimated molecular weights of 110 and 130 kD have been reported(Neufeld and Gospodarowicz, J. Biol. Chem., 260;13860-13868 (1985);Neufeld and Gospodarowicz, J. Biol. Chem., 261:5631-5637 (1986), both ofwhich are incorporated herein by reference). Both receptor proteins bindbasic FGF and acidic FGF, although it appears that the larger bindsbasic FGF preferentially while the smaller has somewhat higher affinityfor acidic FGF.

In addition to potentially useful proliferative effects, basicFGF-induced mitogenic stimulation may, in some instances, bedetrimental. For example, cell proliferation and angiogenesis are anintegral aspect of tumor growth. Basic FGF is thought to play apathophysiological role, for example, in tumor development, rheumatoidarthritis, proliferative diabetic retinopathies and other complicationsof diabetes.

There thus exists a need for being able to inhibit certain mitogeniceffects of basic FGF within the body which may give rise to pathologicalconditions; however, such inhibition must be accomplished in a way thatdoes nob result in the death of the animal or the infliction ofsubstantial harm thereto. Because of the ubiquitous distribution of FGFtarget cells and presumably FGF receptors throughout the body, it wasfelt that such an objective could not be accomplished, yet the presentinvention satisfies this need.

SUMMARY OF THE INVENTION

The invention provides a conjugate comprising basic FGF or otherpolypeptide reactive with an FGF receptor, and a cytotoxic agent. In oneembodiment, the cytotoxic agent is a ribosome-inactivating protein(RIP), such as, for example, saporin, although other cytotoxic agentscan also be advantageously used. The cytotoxic agent can be attached tobasic FGF through a chemical bond or the composition can be prepared asa chimera, using techniques of recombinant DNA. In both cases, theconjugate molecule is designed and produced in such a way that thereceptor-binding epitope of the basic FGF moiety of the complex is leftavailable for recognition by the FGF receptor.

The conjugate can be used to treat FGF-mediated pathophysiologicalconditions by specifically targeting to cells having FGF receptors andinhibiting proliferation of or causing death of the cells. Suchpathophysiological conditions include, for example, tumor development,Dupuytren's Contracture, certain complications of diabetes such asproliferative diabetic retinopathies, and rheumatoid arthritis. Thetreatment is effected by administering a therapeutically effectiveamount of the FGF conjugate, for example, in a physiologicallyacceptable excipient. Additionally, the conjugate can be used to targetcytotoxic agents into cells having FGF receptors, and to inhibit theproliferation of such cells. A method of purifying the conjugate on aheparin-immobilized column is also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a heparin Sepharose chromatography of the conjugationreaction mixture.

FIGS. 2A and 2B show the RIP and binding activities of the basic FGF/SAPconjugate. The activity was compared to SAP alone in a cell-free proteinsynthesis inhibition assay (FIG. 2A) (SAP ▪, basic FGF-SAP ) and thereceptor binding activity was compared to basic FGF in the BHKradioreceptor assay (FIG. 2B) (basic FGF □, basic FGF-SAP ). Each pointis the mean of 3 replicates. Standard deviations were less than 10%.

FIG. 3 shows the effect of basic FGF/SAP on BHK cell proliferation. Cellcounts were normalized to media controls (190,000±15,000). Cell numberwith 10⁻⁸ M of the mitotoxin was 9,527±980. N=3 in all instances. (BasicFGF-SAP , SAP ▪, basic FGF □, basic FGF+SAP ◯.)

FIG. 4 shows the effect of exogenous basic FGF and NGF on cytotoxicity.Basic FGF-SAP was used at a concentration of 10⁻¹⁰ M basic FGF-AP and C:preincubation with equimolar free of basic FGF; D: 10-fold excess offree basic FGF; E: 100-fold excess of basic FGF; F: 1000-fold excess ofbasic FGF; G: equimolar incubation with equimolar free NGF; H: 10-foldmolar excess; I: 100-fold molar excess, J: 100-fold molar excess.

FIG. 5 shows the relationship between toxicity of basic FGF-SAP and FGFreceptor number, determined for each cell line after 48 or 72 hoursexposure to basic FGF-SAP. Cell numbers were determined and theconcentration that reduced the number of cells by 50% was plottedagainst receptor number for that cell line. Receptor number wasdetermined by the method of Moscatelli et al., supra.

FIG. 6 shows the effect of basic FGF-SAP on Dupuytren's Cells asdescribed in Example IV.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a conjugate comprising FGF, orpolypeptide fragments thereof, reactive with an FGF receptor and acytotoxic agent, which composition is effective for inhibiting growthand proliferation of cells having FGF receptors. The composition can beused to counteract the mitogenic effects of basic FGF, where such aneffect is deleterious, such as in tumor angiogenesis and proliferativecomplications of diabetes, such as proliferative retinopathies.

As used herein, the term "FGF" refers to both basic FGF (bFGF) andacidic FGF (aFGF) and other proteins, or fragments thereof, exhibitingFGF mitogenic activity mediated through binding to an FGF receptor. Forexample, a basic FGF peptide having a molecular weight of about 16 kDand a pI of about 9.6 has been described by Esch et al. Other FGFproteins include other forms of basic FGF which have an amino terminalextension, aFGF, hst oncogene, int-2 oncogene, FGF-5, and FGF-6. (SeeBaird et al., Brit. Med. Bull., 45:438-452 (1989)). FGF fragmentsinclude FGF peptides of between about 5-100, preferably about 5-50, morepreferably about 5-25 amino acids that are able to bind to an FGFreceptor. See Baird et al., PNAS, 85:2324-2328 (1988), which isincorporated herein by reference, for an example of basic FGF peptidefragments that are reactive with an FGF receptor. Conjugates of acidicfibroblast growth factor fused to several mutant forms of Pseudomonasexotoxin have been shown to be cytotoxic to a variety of tumor celllines including hepatocellular, prostatic, colon, and breast carcinomas(Siegall et al., The FASEB Journal, 5:2843-2849, October 1991).

FGF expresses mitogenic activity in a wide variety of normal diploidmesoderm-derived and neural crest-derived cells. A test of such "FGFmitogenic activity" is the ability to stimulate proliferation ofcultured bovine aortic endothelial cells, as described in Gospodarowiczet al., J. Biol. Chem., 257:12266-12278 (1982); Gospodarowicz et al.,Proc. Natl. Acad. Sci. USA, 73:4120-4124 (1976), which are incorporatedherein by reference.

The term FGF is used to refer both to proteins having amino acidsequences found in a mammalian host, as well as to modified sequences,having amino acid substitutions, deletions, insertions or additions,which still express mitogenic activity, mediated through binding to anFGF receptor. Purified preparations of basic FGF and acidic FGF arefrequently observed to include several molecular forms of the mitogens.It is understood that differences in amino acid sequences can occur inFGF from different species as well as between FGF from individualorganisms of a particular species. The term is intended to refer to bothproteins isolated from natural sources as well as those madesynthetically, as by chemical synthesis or recombinant means.

The amino acid sequence of an exemplary mammalian basic FGF derived frombovine pituitary tissue is provided in Esch et al., Proc. Natl. Acad.Sci. USA, 82:6507-6511 (1985), which is incorporated herein byreference. As used herein, the term "basic FGF" refers to proteins orpolypeptides having substantially the same amino acid sequence andmitogenic activity as that of the basic FGF described in Esch, supra.cDNAs encoding human aFGF (Jaye et al., Science, 233:541-545 (1986)) andbovine (Abraham et al., Science, 233:545-548 (1986), human (Abraham etal., EMBO J., 5:2523-2528 (1986); Abraham et al., Quant. Biol.,51:657-668 (1986)), and rat (Shimasaki et al., Biochem. Biophys. Res.Commun. (1988); Kurokawa et al., Nucleic Acids Res., 16:5201 (1988))basic FGF have been cloned, and sequenced and predict the existence ofproteins identical to those found by protein sequencing.

As used herein, the term "FGF receptors" refers to receptors which areable to bind basic FGF and transport it into the cell. Included amongthese are the receptors described in Imamura, supra and Moscatelli,supra. As used herein, the term "polypeptide reactive with the FGFreceptor" refers to any polypeptide which is capable of binding an FGFreceptor and of being transported into the cell thereby.

Basic FGF is commercially available, for example, from Amgen (ThousandOaks, Calif.). Acidic FGF is also commercially available from Promega(Madison, Wis.). Basic and acidic FGF can be obtained from a variety oftissue types of mammals. For example, methods of purifying basic FGFusing reverse-phase high performance liquid chromatography (RP-HPLC),heparin-Sepharose affinity chromatography and cation exchange HPLC andRP-HPLC are described in U.S. Pat. No. 4,785,079, as well asGospodarowicz et al., Proc. Natl. Acad. Sci., 81:6963-6967 (1984) andGospodarowicz, Meth. Enzym., 147:106-119 (1987), which are incorporatedherein by reference. In addition, basic FGF can be synthesized, as bychemical or recombinant methods. Expression of a recombinant protein inyeast and E. coli is described in Barr et al., J. Biol. Chem.,263:16471-16478 (1988), which is incorporated herein by reference.

The FGF-cytotoxic agent conjugate can be purified on a column containingimmobilized heparin. Appropriate columns include heparin-Sepharose andheparin-agarose. The bound conjugate can be eluted with a gradient salt,such as NaCl and is eluted between 1 and 3M.

According to one aspect of the invention, basic FGF is conjugated to acytotoxic agent so as to target the cytotoxic agent specifically tocells which exhibit FGF receptors. As used herein, the term cytotoxicagent refers to a molecule capable of inhibiting cell function. The termincludes agents which are only toxic when transported into the cell andalso those whose toxic effect is mediated at the cell surface. A varietyof cytotoxic agents can be used including those which inhibit proteinsynthesis.

In one aspect of the invention, FGF is combined with aribosome-inactivating protein (RIP) such as, for example, the type-1 RIPsaporin-6 (SAP) or other SAP derivatives. SAP is a potent RIP which isisolated from the seeds of the plant Saponaria officinalis (see Stirpe,et al., Biochem. J., 216:617-625 (1983)). Other suitable RIPs include,but are not limited to, ricin, ricin A chain, gelonin, diphtheria toxin,diphtheria toxin A chain, trichosanthin, tritin, pokeweed antiviralprotein (PAP), mirabilis antiviral protein (MAP), Dianthins 32 and 30,abrin, monordin, bryodin, and shiga. L. Barbieri et al., Cancer Surveys,1, 489-520 (1982) and EPO published patent application No. 466,222,incorporated herein by reference, provide lists of numerous RIPs andtheir sources.

Other cytotoxic agents which are considered to be functionallyequivalent to the aforementioned RIPs include Pseudomonas exotoxin andmetabolic inhibitors which are known in this art, but are not limitedthereto. Therefore, the term RIPs is used in this application to broadlyinclude such cytotoxins. For example, chimeric proteins composed ofacidic fibroblast growth factor fused to several mutant forms ofPseudomonas exotoxin have been produced that have proven to be cytotoxicto a variety of tumor cell lines, including hepatocellular, prostatic,colon, and breast carcinomas (Siegall et al., The FASEB Journal,5:2843-2849, October 1991). The pseudomonas toxin has also been shown tobe effective at killing cells expressing epidermal growth factorreceptors when fused to transforming growth factor type α as a chimericprotein. Chaudhary et al., PNAS, 84:4538-4542, (1987).

In another aspect of the invention, the cytotoxic agent is a drug.Examples of such drugs are anthracyclines such as the daunomycins(including daunorubicin and doxorubicin) and methotrexate and itsanalogs. Others are known to those skilled in the art.

FGF can be conjugated to a protein cytotoxic agent by any means known tothose skilled in the art, such as through derivitization with a reactivesulfhydryl containing moiety such as SPDP, or via a suitable crosslinking agent, such as glutaraldehyde or carbodiimide. In oneembodiment, the cytotoxic agent is derivatized with a reactivesulfhydryl containing agent, such asN-succinimidyl-3(2-pyridyldithio)propionate. FGF is then added to andmixed with the derivatized cytotoxic agent. The FGF conjugate can beseparated from the unreacted products on a column. Alternatively, FGFcan be conjugated to a drug, such as 14 bromo doxorubicin through thesugar moiety, as by the cis-aconitate method (Shen and Riser, BBRC,102:1048 (1981), which is incorporated herein by reference).

Alternatively, chimeric FGF-conjugates can be prepared by recombinantmethods. Such methods as applied to conjugates of IL-2 or TGFα areprovided in Chaudhary et al., Proc. Natl. Acad. Sci. USA, 84:4538-4542(1987) and Lorberman-Galski et al., Proc. Natl. Acad. Sci. USA,85:1922-1926 (1988), which are incorporated herein by reference. Seealso, Maniatis, et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory (1982), which is incorporated herein byreference.

A conjugate containing FGF and a cytotoxic agent is useful in treating avariety of FGF-mediated pathophysiological conditions. As used herein,the term "FGF-mediated pathophysiological condition" refers to adeleterious condition characterized by or caused by proliferation ofcells which are sensitive to basic FGF mitogenic stimulation. BasicFGF-mediated pathophysiological conditions include, but are not limitedto, tumors, rheumatoid arthritis, Dupuytren's Contracture and certaincomplications of diabetes such as proliferative retinopathy.

FGF-cytotoxic agent conjugates can be used to target the cytotoxic agentto cells expressing FGF receptors in order to cause cell death.Surprisingly, there is a direct relationship between the number of FGFreceptors per cell and the dose at which 50% of the cells are killed(the ED₅₀), as is shown in FIG. 5. Moreover, for cells with extremelyhigh receptor numbers, for example, BHK cells, the ED₅₀ is identical tothe affinity constant of basic FGF for its receptor (both are about 25pM for BHK cells). This unexpected result indicates that the presence ofthe cytotoxic agent, even such a large molecule as SAP, does not reducebasic FGF activity. Moreover, these results indicate that these cellsthat are expressing a large number of basic FGF receptors areparticularly sensitive to the conjugate.

In order to treat FGF-mediated pathophysiological conditions, atherapeutically effective amount of FGF-cytotoxic agent conjugate isadministered to a mammal in a physiologically acceptable excipient.Examples of physiologically acceptable excipients include PBS andsaline.

The following examples are intended to illustrate but not limit theinvention.

EXAMPLE I CONJUGATION OF FGF WITH SAPORIN

Recombinant basic FGF corresponding to the sequence of 154 amino acids(Abraham et al., Quant. Biol., 51:657-668 (1986), which is incorporatedherein by reference) was obtained from Farmitalia Carlo Erba. Saporin-6was purified according to the method of Stirpe, et al., supra, asmodified by Lappi, et al., Biochem. Biophys, Res, Comm., 129:934-942(1985), which is incorporated herein by reference. Briefly, seeds ofSaponaria officinalis were extracted by grinding in 0.14M NaCl in 5 mMsodium phosphate buffer, pH 7.2 (8 ml/g). After overnight stirring at 4°C., extracts were strained through cheese-cloth and were centrifuged at28000 g for 30 minutes. The supernatant was separated from the sedimentand from floating fat, and is referred to as "crude extract."

Crude extracts were dialyzed against 5 mM sodium phosphate buffer, pH6.5, centrifuged at 28000 g for 30 minutes and applied to a CM cellulosecolumn (CM 52; Whatman, Maidstone, Kent, U.K.), which after washing, waseluted with a 0-0.3M NaCl gradient in the same buffer. This material wasthen dialyzed against water and chromatographed on an FPLC Mono S column(Pharmacia, Uppsala, Sweden) equilibrated with 50 mM sodium borate pH9.5, 0.156M sodium chloride. The protein was eluted with a 20 minutegradient from 0.156M to 0.186M sodium chloride. The resultant peakmaterial was then extensively dialyzed against Milli-Q water (Millipore,Bedford, Mass.). A portion of the dried material was weighed anddissolved in water at a concentration of 1 mg/ml. An ultravioletspectrum was recorded giving a 1% extinction coefficient of 6.4 at 277nm, the absorbance maximum. At 280 nm the E₂₈₀ was 6.0. A protein assayusing the Lowry method (Lowry, et al., J. Biol. Chem., 193:265-275(1951)) using BSA as a standard gave a result of 1.07 mg/ml.

SAP was derivatized with N-succinimidyl-3(2-pyridyldithio)propionate(SPDP; Pharmacia Fine Chemicals, Piscataway, N.J.) according to themanufacturer's instructions. Briefly, SAP was dissolved in (2.7 mg/ML)sodium phosphate buffer (0.1M, pH 7.5) containing NaCl (0.1M). A 1.25molar excess of SPDP, dissolved in ethanol, was added drop by drop whilestirring, and allowed to react for 30 minutes at 23° C. with occasionalstirring. Excess reagent and low molecular weight reaction products wereremoved by gel filtration. Basic FGF (2 mg/ml) was added to and mixedwith the derivatized saporin (6 mg/ml in 0.1M sodium phosphate, 0.1Msodium chloride, pH 7.5) for two hours at room temperature. The reactionwas terminated by the addition of 35 μL of 0.1M iodacetamide. After anadditional 30 minutes, the reaction mixture was diluted to 30 ml andloaded onto a heparin-Sepharose (Pharmacia) column (0.5×5.5 cm). Thebound proteins were eluted with a step gradient of 0.6M, 1M and 2M NaClin 10 mMTRIS, pH 7.4. The material eluting between 1M and 2M was pooled.Final purification of the conjugate was achieved after the pool wasdialyzed against water and chromatographed on a Mono S 5/5 NaCl cationexchange column (Pharmacia) (buffer A: 50 mM sodium borate, pH 8.0,buffer B: 0.5M NaCl in buffer A). Fractions containing the conjugatewere detected by silver staining after PhastGel (Pharmacia)electrophoresis and appropriate fractions were pooled for analysis.

Synthesis of the conjugate was assessed by gel electrophoresis andallowed to proceed until no detectable basic FGF remained in thereaction mixture. Chromatography on heparin-Sepharose (FIG. 1) andsubsequent electrophoretic analysis of each of the peak fractions showedthat, while SAP does not bind to heparin-Sepharose, the conjugate does.Only small amounts of the conjugate were released during the 1.0M NaClwash. The major product eluted with the 2M wash and contained equimolaramounts of SAP and basic FGF (Mr˜40,000). However, there was also aportion of the conjugate that has an estimated Mr>68,000 presumably as aresult of the conjugation of two molecules of basic FGF per molecule ofsaporin.

Unambiguous identification of the SAP-basic FGF conjugate wasaccomplished using sequence specific antisera raised in rabbits. Theimmunogen used was a fragment of basic FGF comprising amino acids 1through 24, chemically synthesized using a 990 Peptide Synthesizer(Beckman Instruments, Brea, Calif.). Western blot analysis showed thatall molecular weight forms of the conjugate contained both basic FGF andSAP. The antiserum recognizes the midportion of the peptide andcross-reacts on equimolar basis with purified bovine and recombinanthuman basic FGF.

Samples in a sodium dodecyl sulfate-containing polyacrylamide gel, afterelectrophoresis, were electroblotted onto nitrocellulose membranes, andallowed to air dry. The membrane was covered with TRIS-buffered saline(TBS) and agitated for 10 minutes. The solution was aspirated anddiscarded. The membrane was covered with 5% nonfat milk (NFM) in TBS andagitated for 10 minutes. The solution was aspirated and discarded.Primary antibody, either anti-SAP or anti-basic FGF anti-serum, at aconcentration of 1/100 in NFM/TBS was added and agitated overnight. Thesolution was aspirated and discarded. The membrane was covered with TBS,agitated for 10 minutes and the solution aspirated and discarded. Themembrane was covered with 0.05% NP40/TBS and shaken 1 minute; thesolution was aspirated and discarded. The final TBS and NP40/TBS washeswere replated twice. Horseradish peroxidase labelled anti-IgG at adilution of 1/2000 in NFM/TBS was added and the membrane agitated for 2hours. The TBS and NP40/TBS wash steps were repeated. The membrane wasplaced in a solution (Freshly mixed) of 60 mg 4-chloro-1-naphthol in 20mL methanol and 100 mL double distilled water and 10 μL 30% H₂ O₂ andallowed to develop. The solution was aspirated and discarded and thereaction stopped by rinsing with water. The membrane was allowed to dry.

EXAMPLE II ACTIVITY OF THE FGF-SAP CONJUGATE

The capacity of the conjugate to recognize the basic FGF receptor wasexamined in BHK cells using the procedure described by Moscatelli, etal., J. Cell Physiol., 131:123-130 (1987), which is incorporated hereinby reference. Briefly, cells were grown to subconfluence and incubatedin 300 μL buffer containing F-12 14 mM NaHCO₃, 25 mM HEPES and 0.2%gelatin at 4° C. for two hours with 10 μL radioiodinated basic FGF inthe presence of various concentrations of basic FGF or the conjugate.The cells were then washed three times with 0.5 mL phosphate-bufferedsaline (PBS), and twice with 2M NaCl in PBS. Binding to the highaffinity receptor was determined by counting the membrane fraction thatwas solubilized 0.5% Triton X-100 in PBS (pH 8.1).

The protein synthesis inhibition activity of the SAP protein wascompared to the protein synthesis inhibition activity of the basicFGF-SAP conjugate in in vitro assays of protein synthesis as describedin Siehn et al., Blood, 72:756-765 (1988), which is incorporated hereinby reference. The cytotoxic activity of the conjugate was tested on babyhamster kidney fibroblasts (ATCC Accession No. CRL 6281). BHK cells wereplated in 24 well plates at a concentration of 5000 cells/ml andincubated overnight at 37° C., 5% CO₂. The following morning,HEPES-buffered DMEM and F-12 media (1:1) plus 5% FCS was aspirated fromthe wells and replaced with media alone or with media containing theconjugate, basic FGF or saporin. Two days later, the cells were washedtwice, trypsinized and cell number determined with a Coulter ParticleCounter (Coulter Electronics, Hialeah, Fla.) .

As shown in FIG. 2A, the conjugate retains saporin activity when testedin an in vitro protein synthesis inhibition assay. The conjugate, asexpected, is slightly less active (about two-fold) than free SAP. Thisis consistent with the low level of derivatization of SAP prior to theconjugation of (0.8 moles SPDP/mole) and with probably steric hindrancedue to the presence of bound basic FGF. In contrast, the resultsobtained in the radioreceptor assays for basic FGF (FIG. 2B) showed thatthe basic FGF-SAP is equipotent to, if not slightly more active than,basic FGF in the binding assay. Thus, it appears that the commitment offree sulfhydryl groups in basic FGF to bridging with SAP does notinterfere with its capacity to recognize its receptor. If anything, thisreaction may be stabilizing basic FGF.

Basic FGF-SAP is a potent cytotoxic factor for BHK cells (FIG. 3). SAPhas no toxic effect on these cells even at the highest dose tested (10⁻⁸M) and basic FGF alone has a slight inhibitory effect on proliferation.A mixture of basic FGF and SAP had a slight toxicity but only at thehighest concentration tested. The ID₅₀ (25 pM) for the cytotoxic agentcompared well with the potency of basic FGF (15 pM) in proliferationassays. Specificity of the cytotoxic agent was examined in competitionexperiments in an effort to establish that the mitotoxic activity of theconjugate is receptor specific. BHK cells were preincubated for one hourwith various levels of basic FGF or nerve growth factor (NGF) prior totreatment of the cells with the cytotoxic agent. As shown in FIG. 4,there is a dose-related inhibition of the cytotoxic activity in thepresence of increasing amounts of basic FGF. In contrast, athousand-fold excess of NGF has no effect.

EXAMPLE III INHIBITION OF ANGIOGENESIS IN RABBIT CORNEA

Elvax (ethylene-vinyl acetate copolymer resin, Dupont, Wilmington, Del.)pellets were produced in the following manner. About 60 mg of washed anddried Elvax was dissolved in 500 μL of methylene chloride. This wasadded to 50 μg of dried basic FGF. 5 μL drops were dropped onto a slidefrozen in dry ice. Pellets were left in the freezer overnight and thendried in a desiccator.

New Zealand white rabbits were anaesthetized with Innovar Vet: 1 mL/kg.An incision was made in the cornea of the rabbit eye and a pocket wasopened with spatula or forceps. One pellet was inserted in the pocket.Pellets were inserted in both eyes. The eye was washed with saline, and1 ml of gentamicin was injected intramuscularly. The rabbit was left forfive days, and angiogenesis was observed. After five days, each left eyewas treated with 20 μL of 100 ng basic FGF-SAP prepared as in Example Iin 0.25% BSA. The right eyes were treated with 20 μL of 0.25% BSA alone.The treatment was done twice daily by dropping the solution as eye dropsonto the cornea of the rabbit. The person treating the animals wasunaware of the identity of the samples. After 10 days, the animals wereevaluated for angiogenesis of the cornea by microscopic analysis by anevaluator who did not know the treatment regimen. Angiogenesis wasjudged, with +++ as being maximal anglogenesis and - as being noangiogenesis.

The results are provided in Table I. As can be seen, anglogenesis incorneas treated with basic FGF-SAP was markedly reduced over that ofcontrols.

                  TABLE I                                                         ______________________________________                                        ANIMAL        RIGHT EYE LEFT EYE                                              ______________________________________                                        995           +         -                                                     997           +++       +                                                     998           +++       +                                                     999           ++        -                                                     ______________________________________                                    

EXAMPLE IV EFFECT OF FGF-SAP IN DUPUYTREN'S CELL

Cells obtained from surgical removal of tissue from the hand of adultpatients diagnosed as having Dupuytren's Contracture, a malady effectingmovement of the hand, were placed in primary culture. These cells havebetween 10,000 and 15,000 basic FGF receptors per cell.

The cells were grown overnight in a 24-well tissue culture dish at aconcentration of 10,000 cells per well in HEPES-buffered DMEM with 10%FCS. The next morning the media was removed and replaced with mediacontaining concentrations of basic FGF-SAP conjugate ranging from 10⁻⁸to 10⁻¹² molar. Controls included wells treated with media only, andwells treated with similar concentrations of basic FGF alone, saporinalone, and basic FGF and saporin together but not conjugated. The cellswere returned to the incubator for 72 hours. At the end of thisincubation, the cells were washed, removed with trypsin and counted on aCoulter cell counter. The number of cells in the media controls wascompared with the number of cells in the treated wells (as describedabove). The results of these cell killing assays are shown in FIG. 6. Ascan be seen, Dupytren's cells are sensitive to basic FGF-SAP. Similarresults were obtained with three other cell samples.

Pseudomonas exotoxin (PE) is substituted for saporin in the protocol ofExample I, derivatized with SPDP and conjugated with basic FGF. ThebFGF-PE conjugate is employed as described in Examples II, III, and IVfor bFGF-SAP and similar results are obtained.

In addition, acidic FGF is substituted for basic FGF in the Example Iprotocol and mixed with the derivatized saporin; the conjugate ispurified as generally described in Example I. The aFGF-SAP conjugate isemployed as described in Examples II, III, and IV for bFGF-SAP andsimilar results are obtained. The peptide FGF-(93-120)-NH₂ (amino acids93-120 of basic FGF having 146 amino acid residues, see U.S. Pat. No.5,132,408) is substituted for basic FGF in the above protocol and mixedwith the derivatized saporin; the conjugate is purified as generallydescribed in Example I. The FGF-(93-120)-SAP conjugate is employed asdescribed in Examples II, III, and IV for bFGF-SAP and similar resultsare obtained.

Although the invention has been described with reference to thepresently-preferred embodiments, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

We claim:
 1. A method of treating an FGF-mediated pathophysiologicalcondition, comprising administering a therapeutically effective amountof a conjugate comprising a cytotoxic agent and a polypeptide reactivewith a FGF receptor wherein the polypeptide reactive with the receptoris selected from the group of polypeptides consisting of polypeptidesthat exhibit mitogenic activity mediated through binding to an FGFreceptor and fragments of polypeptides that exhibit FGF mitogenicactivity mediated through binding to an FGF receptor and that bind to anFGF receptor and are transported into the cell, thereby internalizingthe linked cytotoxic agent.
 2. The method of claim 1 wherein saidpolypeptide reactive with an FGF receptor is basic FGF.
 3. The method ofclaim 2 wherein said cytotoxic agent is a ribosome-inactivating protein.4. The method of claim 1 wherein said cytotoxic agent is saporin.
 5. Themethod of claim 1 wherein said cytotoxic agent is selected from thegroup consisting of methotrexate, anthracyclines and Pseudomonasexotoxin.
 6. A method of inhibiting proliferation of cells having FGFreceptors, comprising administering to said cells an effective amount ofa conjugate comprising a cytotoxic agent and a polypeptide reactive witha FGF receptor wherein the polypeptide reactive with the receptor isselected from the group of polypeptides consisting of polypeptides thatexhibit mitogenic activity mediated through binding to an FGF receptorand fragments of polypeptides that exhibit FGF mitogenic activitymediated through binding to an FGF receptor and that bind to an FGFreceptor and are transported into the cell, thereby internalizing thelinked cytotoxic agent.